Malaria caused by infection with Plasmodium falciparum exerts an enormous toll on human health in tropical regions. Current antimalarial treatments are being severely compromised by the spread of P. falciparum strains resistant to chloroquine (that interferes with heme detoxification) and pyrimethamine-sulfadoxine (that inhibits folate biosynthesis). Several groups have recently defmed P. falciparum type II fatty acid synthesis (FAS-II), an apicoplast pathway that is lacking in humans, as a unique drug target. The FAS-II enzyme enoyl ACP reductase (ENR) is the target of several widely used antimicrobial agents including triclosan, isoniazid and ethionamide. Our Program, representing a public/private partnership between GlaxoSmithKline, Texas A&M University and the Albert Einstein College of Medicine, proposes to implement a high throughput screen against purified P. falciparum ENR (PfENR) and follow this with a Hits to Leads program that integrates chemistry, biochemistry, structural biology, molecular biology and parasitology. In Aim 1 of this project, we will evaluate the efficacy of test ENR inhibitors against drug-resistant and drug-sensitive P. falciparum in vitro. Aim 2 will assess the in vivo efficacy of promising PfENR inhibitors, using non-transformed P. berghei as well as transgenic P. berghei clones expressing the enoyl ACP reductase from P. falciparum or P. vivax (the second most important human malaria species) in the place of the rodent malarial enzyme (the transgenic P. berghei lines will be generated as part of Project 3). Aim 3 proposes to develop transgenic, "mode of action" P. falciparum lines that underexpress PfENR or are functionally complemented for ENR activity, to assess inhibitor specificity. Aim 4 will develop a transgenic P. falciparum line expressing the P. vivax enr in the place of pfenr, as a surrogate model to measure in vitro activity against P. vivax. This project provides key resources and reagents to identify potent, orally bioavailable inhibitors that can move down the critical path and enter preclinical development as new candidate antimalarial drugs.